In the production of proteins or polypeptides from cultures of microorganisms or cell lines, the final production step is the recovery and optionally the concentration of the product of interest. Culture media in which the cells have been grown and which contain secreted proteins, and, in particular, cell lysates containing intracellular proteins of interest also contain, to a greater or lesser extent, other proteins produced by the cells, apart from other contaminants, such as media components, nucleic acids and the like. In order to obtain a purified protein product, it is therefore necessary to separate the protein of interest from other proteins and polypeptides in the crude material containing this protein.
One widely practiced method of ensuring such a separation is affinity chromatography which generally involves a specific interaction between an insoluble immobilized ligand and a soluble protein, cf. A. Johnstone and R. Thorpe, Immunochemistry in Practice, 2nd Ed., Blackwell Scientific Publications, 1987, pp. 207-240. By interacting with the ligand, the protein is temporarily rendered insoluble and is retained on the solid support on which the ligand is immobilized while the soluble contaminants are eluted. The binding of the protein to the ligand conventionally takes place in an aqueous buffer at a neutral pH. The protein is subsequently released from the immobilized ligand by a change in the elution conditions, such as a jump in the pH, an increase in temperature or elution with a denaturing agent, an organic solvent or an unphysiologically high concentration of a salt. As a result of these procedures, the protein is often recovered in denatured form and will have to be subjected to further procedures in order to become available in its native conformation.
Examples of commonly used ligands are antibodies, in particular monoclonal antibodies, which can be made to be more selective and to bind more firmly most other known ligand and which are therefore preferred as they result in a higher purity of the eluted protein product. In order to obtain an antibody in sufficient quantities, however, the protein to be purified should usually be available in substantially pure form for the immunization procedure. This approach to solve the problem therefore implies that the problem of protein recovery has already been solved by other means.
The amino acid sequence, i.e. primary structure, of many proteins may initially be arrived at by deduction from the DNA sequence of their corresponding genes. This procedure often takes place before the protein product itself has been isolated. Knowledge of the amino acid sequence of a protein makes it possible to synthesize peptide fragments thereof, e.g. by solid phase peptide synthesis techniques. Such peptide fragments may then be used to raise antibodies against the protein to be purified. However, it has often been found that the thus generated antibodies are reactive with the synthetic peptide fragment against which they have been raised, but not with the native protein of which the peptide constitutes a fragment. This phenomenon is thought to be ascribable to sterical hindrance or to differences in conformation between the synthetic peptide and the native protein, the peptide fragment having a highly flexible conformation in which side chains are exposed, whereas the same fragment is less flexible in the entire protein and has a number of its side chains, in particular hydrophobic side chains, embedded in the interior of the protein. Whether or not this is the correct explanation, it is at any rate often the case that antibodies raised against synthetic peptide fragments of a native protein do not bind the entire native protein. Similar results are also often obtained when denatured proteins are used as immunogens.